With characterization of genetic defects in severe genetic skin disease, such as laminin 5 chans in the majority of patients with severe junctional epidermolysis bullosa (JEB), the time is ripe for cutaneous genetic therapy. Important for the reliability and success of gene therapy efforts is the ability to control therapeutic gene delivery and current gene therapy efforts have been plagued by an inability to maintain corrective gene expression. This loss of gene expression has been a recurrent stumbling block in experimental gene delivery efforts in the skin and may result from a number of factors, including inactivation of viral promoters and loss of therapeutic genetic epements. We plan, therefore, to develop the ability to achieve high efficiency, sustained target gene expression in human keratinocytes as a platform for optimized human gene therapy for JEB. Because promoters and other features of gene regulation exhibit vast differences in activity in different tissue cell types, we will develop these capabilities specifically for keratinocytes because they are the main producers of laminin 5. First, we will develop the capability for sustainable gene expression in keratinocytes in vitro. To do this, we will generate new retroviral gene delivery vehicles utilizing native promoters and insulating elements and will test the resultant vectors for sustainable gene expression in vitro. Second, we will develop an approach to achieve sustainable keratinocyte gene expression in vivo using the human skin/SCID mouse xenograft model. Insulated super-high titer amphotropic retroviral expression vectors previously refined in vitro will be utilized to generate engineered human epidermis on SCID mice and the duration of target gene expression in vivo determined. Optimized sustainable vehicles will then be used to produce laminin 5 chain delivery vectors. In a future aim we will produce vectors for regulated delivery of the laminin 5 alpha chain in an effort to investigate therapeutic gene dosage requirements for JEB and to allow development of approaches to regulated overexpression of the entire laminin 5 heterotrimer in keratinocytes. At the end of this proposed granting period, we hope to have developed a general approach to achieve sustainable keratinocyte gene delivery and to have applied these capabilities in the development of an effective approach to sustainable therapeutic gene delivery for JEB in vivo.